Uncoupling protein 2 (UCP2) (Fleury, C., et al., Nat. Genet, 15:269 (1997); Gimeno, R. E., et al., Diabetes, 46:900 (1997)) and uncoupling protein 3 (UCP3) (Boss, O., et al., FEBS Lett., 408:39 (1997); Vidal-Puig, A., et al., Biochem. Biophys. Res. Commun., 235:79 (1997); Gong, D. W., et al., J. Biol. Chem. 272:24129 (1997)) are recently discovered members of the mitochondrial inner membrane carrier family with high homology to UCP1 (Nicholls, D. G., et al., Physiol. Rev., 64:1 (1984); Klingenberg, M., and Huang, S. G., Biochim. Biophys. Acta., 1415:271 (1999)) and expression patterns which are consistent with the hypothesis that they play a role in the regulation of cellular processes in which ATP plays a regulatory function. Consistent with this theory, studies in which UCP2 and UCP3 have been overexpressed in yeast (Rial, E., et al., EMBO J, 18:5827 (1999); Hinz, W., et al., FEBS Lett, 448:57 (1999); C. Y Zhang, et al., FEBS Lett, 449:129 (1999)) or reconstituted into proteoliposomes (Jaburek, M., et al., J Biol. Chem., 274:26003 (1999)) indicate a proton leak (and as a consequence modulator of ATP) role for these new UCPs. UCP3 is expressed primarily in skeletal muscle where it likely plays a role in regulated thermogenesis. In contrast, UCP2 has a nearly ubiquitous expression pattern, but at varying levels in a number of tissues and cell types including tissues involved in glucose homeostasis (pancreatic islets, white fat, brown fat, heart, skeletal muscle). For example, UCP2 mRNA (Zhou, Y. T., et al., Proc. Natl. Acad. Sci. U.S.A., 94:6386 (1997); Chan, C. B., et al., Diabetes 48:1482 (1999)) and protein are highly expressed in pancreatic xcex2-cells.
xcex2-cell function deteriorates in many individuals with obesity and insulin resistance, culminating in the development of type II diabetes mellitus. UCP2 mRNA expression is increased in adipose tissue of ob/ob obese mice, raising the possibility that it may also be increased in xcex2-cells as well. If true, obesity-induced UCP2 expression in xcex2-cells could contribute to xcex2-cell dysfunction, promoting the development of diabetes. Consistent with this theory, it has been reported that UCP2 lies within a major quantitative trait loci (QTL) (murine chromosome 7; rat chromosome 1 and human chromosome 11) controlling diet-induced hyperinsulinemia in C57B1/6 mice (Fleury, C., et al., Nat. Genet, 15:269 (1997); Seldin, M. F., et al., J. Clin. Invest., 94:269 (1994)); glucose intolerance and adiposity in the GK (Goto-Kakizaki) model of type 2 diabetes the rat (Gauguier, D., et al., Nat. genet., 12:38 (1996); Galli, J., et al., Nat. genet., 12:31 (1996); Kaisaki, P. J., et al., Mamm. genome, 9:910 (1998)), and human insulin-dependent diabetes locus-4 (Fleury, C., et al., Nat. Genet, 15(3):269-272 (1997)). Unfortunately, little is known about regulation of xcex2-cell UCP2 gene expression during the pathogenesis of NIDDM. Similarly, little is known about regulators of UCP2 activity, which could also be altered, contributing to xcex2-cell dysfunction.
Thus, additional studies, particularly in vivo studies, are needed to elucidate the biochemical physiological functions of UCP2 and to identify molecular targets, regulators and therapeutic strategies for the treatment or prevention of diseases or conditions associated with UCP2-regulated cellular processes.
The present invention relates to a transgenic non-human mammal (e.g., mouse) which lacks a functional UCP2 gene (also referred to herein as transgenic non-human UCP2 knockout mammal or a UCP2 knockout mammal). The transgenic non-human mammal of the present invention can have at least one non-functional allele for the UCP2 gene. In one embodiment, the transgenic non-human mammal is characterized by a disruption of the UCP2 gene which is either a homozygous disruption or a heterozygous disruption. In a particular embodiment, the genome of the UCP2 knockout mammal comprises a disruption of a segment between introns 2 and 7 of the UCP2 gene. In another embodiment, the genome of the UCP2 knockout mammal comprises an insertion of an exogenous nucleic acid sequence into an exon of the UCP2 gene.
As a result of the disruption of the UCP2 gene, the transgenic knockout mammal of the present invention manifests a particular phenotype. In one embodiment, the UCP2 knockout mammal has altered insulin/glucose homeostasis. In a particular embodiment, the transgenic non-human mammal is characterized by increased glucose-stimulated insulin secretion.
The invention further provides a method of producing a transgenic non-human mammal which lacks a functional UCP2 gene. In this method, a targeting vector is introduced into an embryonic stem cell to produce a transgenic stem cell in which the UCP2 gene is disrupted. A transgenic embryonic stem cell which includes a disrupted UCP2 gene due to the integration of the targeting vector into its genome is then selected. The selected embryonic stem cell is introduced into a blastocyst, thereby forming a chimeric blastocyst; and the chimeric blastocyst is introduced into the uterus of a pseudopregnant mammal wherein the pseudopregnant mammal gives birth to a transgenic non-human mammal which lacks a functional UCP2 gene due to heterozygous disruption of the UCP2 gene. The method can further comprise breeding the transgenic non-human mammal which lacks a functional UCP2 gene due to a heterozygous disruption with a second mammal of the same species to generate F1 progeny having a heterozygous disruption of the UCP2 gene, thereby expanding the population of mammals having a heterozygous disruption of the UCP2 gene. The F1 progeny are then crossbred to produce a transgenic non-human mammal which lacks a functional UCP2 gene due to a homozygous disruption of the UCP2 gene.
The present invention also relates to constructs or vectors (e.g., UCP2 targeting construct) designed to disrupt the function of a wild type mammalian UCP2 gene. In one embodiment, the invention provides a construct which comprises about 5.5 kb of a UCP2 sequence which is 5xe2x80x2 of an expression cassette. In a particular embodiment, the construct comprises 8.7 kb of genomic UCP2 sequence wherein 5.5 kb of the 8.7 kb genomic sequence is 5xe2x80x2 of an expression cassette and 3.2 kb of the 8.7 kb genomic sequence is 3xe2x80x2 of the expression cassette. More specifically, the invention provides a UCP2 gene replacement vector in which the genomic nucleotide sequence of the UCP2 gene between introns 2 and 7 is removed and/or replaced with a PGK-Neo-Poly(A) expression cassette.
The present invention also provides cells, cell lines, mammalian tissues, cellular extracts, organelles (e.g., mitochondria) and organs which lack a functional UCP2 gene. In one embodiment, the cells are pancreatic beta cells.
The cells, cell lines, mammalian tissues, cellular extracts, organelles (e.g., mitochondria) and organs of the instant invention can be used in a method for determining whether an agent inhibits UCP2. For example, pancreatic tissue, islets or cells isolated from wild-type mouse and a UCP2 knockout mouse can be used in combination to identify an agent which inhibits UCP2-mediated negative regulation of xcex2-cell secretion of insulin. In one embodiment, a suitable in vitro screening method comprises combining cells which comprise a wild type UCP2 gene (wild type cells), an amount of glucose sufficient to stimulate insulin production and the agent; and combining cells which lack a functional UCP2 gene (UCP2 knockout cells), an amount of glucose sufficient to stimulate insulin production of the cells and the agent. The cells are maintained under conditions appropriate to stimulate insulin production. The amount of insulin produced by the wild type cells is compared to the amount of insulin produced by the UCP2 knockout cells, wherein if the amount of insulin produced by the wild type cells is increased compared to the amount of insulin produced by the knockout cells, then the agent inhibits UCP2.
The in vitro screening method can further comprise the use of any suitable control. For example, in one embodiment, the in vitro screening method can further comprise combining the wild type cells with an amount of glucose sufficient to stimulate insulin production in the absence of the agent; and combining UCP2 knockout cells with an amount of glucose sufficient to stimulate insulin production in the absence of the agent. The cells are maintained under conditions appropriate to stimulate insulin production. The amount of insulin produced by the wild type cells in the presence of the agent is compared to the amount of insulin produced by the wild type cells in the absence of the agent; and the amount of insulin produced by the UCP2 knockout cells in the presence of the agent is compared to the amount of insulin produced by the UCP2 knockout cells in the absence of the agent. If the amount of insulin produced by the wild type cells in the presence of the agent is increased compared to the amount of insulin produced by the wild type cells in the absence of the agent, and the amount of insulin produced by the UCP2 knockout cells in the presence of the agent is similar to the level produced by the knockout cells in the absence of the agent, then the agent inhibits UCP2. According to the instant invention, an appropriate pancreatic xcex2-cell based assay for the identification of agents which inhibit UCP2-mediated regulation of insulin secretion can be practiced with cells characterized by a disruption of the UCP2 gene selected from the group consisting of: a homozygous disruption and a heterozygous disruption.
Alternatively, a high through-put screening assay for the identification of agents which modulate (e.g., inhibit or activate) UCP2 activity can be established based on the knowledge that UCP2 effects ATP/ADP ratios. For example, cells (or cell lines) comprising a disrupted UCP2 gene which have either been derived from the transgenic non-human UCP2 knockout mammals described herein, or genetically engineered by the gene targeting method described herein can be transfected with a reporter gene, such as a luciferase expression construct designed to emit a luminescence signal that is directly correlated to ATP concentration (Kohler, M., et al., FEBS Lett., 441:97-102 (1998) and Kennedy, H. J., et al., JBC, 274:13281-91 (1999)). The resulting cells can then be contacted with various agents which are being evaluated for their ability to modulate (e.g., inhibit or enhance) UCP2 activity. Modulators of UCP2 activity can be identified by comparing the luminescence signal of cells expressing a wild-type UCP2 with the signal of cells whose genome comprises a disrupted UCP2 gene. A UCP2 specific agent can be determined by identifying agents which modulate the ATP level of cells comprising wild-type UCP2 gene relative to the ATP level of control cells, but which do not effect the ATP level of cells comprising a disrupted UCP2 gene. Based on the knowledge that ATP/ADP ratios control insulin secretion in pancreatic xcex2-cells, it is also reasonable to assume that the class of UCP2-specific agents identified in the above described assay can be used to control insulin secretion.
Another embodiment of the present invention provides an in vivo screening method for determining whether an agent inhibits UCP2. The increased glucose-sensitivity of the transgenic non-human UCP2 knockout mammals of the instant invention can be utilized as the basis of a screening assay in which an agent that is being evaluated for its ability to inhibit the UCP2-mediated negative regulation of insulin production in response to glucose stimulation. In one embodiment, a suitable in vivo screening method comprises administering to a non-human mammal which comprises a wild type UCP2 gene (wild type mammal), an amount of glucose sufficient to stimulate insulin production and the agent; and combining a transgenic non-human mammal which lacks a functional UCP2 gene (UCP2 knockout mammal), an amount of glucose sufficient to stimulate insulin production of the cells and the agent. The amount of insulin produced by the mammals is measured. The amount of insulin produced by the wild type mammal is compared to the amount of insulin produced by the UCP2 knockout mammal. If the amount of insulin produced by the wild type mammal is increased compared to the amount of insulin produced by the knockout mammal, then the agent inhibits UCP2.
The in vivo screening method can further comprise the use of any suitable control. For example, in one embodiment, the in vivo screening method can further comprise administering to a wild type mammal an amount of glucose sufficient to stimulate insulin production in the absence of the agent; and administering to a UCP2 knockout mammal an amount of glucose sufficient to stimulate insulin production in the absence of the agent. The amount of insulin produced by the mammals is measured. The amount of insulin produced by the wild type mammal in the presence of the agent is compared to the amount of insulin produced by the wild type mammal in the absence of the agent; and the amount of insulin produced by the UCP2 knockout mammal in the presence of the agent is compared to the amount of insulin produced by the UCP2 knockout mammal in the absence of the agent. If the amount of insulin produced by the wild type mammal in the presence of the agent is increased compared to the amount of insulin produced by the wild type mammal in the absence of the agent, and the amount of insulin produced by the UCP2 knockout mammal in the presence of the agent is similar to the level produced by the knockout mammal in the absence of the agent, then the agent inhibits UCP2.
Also encompassed by the present invention is an method of identifying an agent which mimics UCP2 activity. In one embodiment, the method comprises introducing the agent (e.g., potential UCP2 mimic) into cells which lack a functional UCP2 gene and determining whether a UCP2-mediated cellular function (one or more) occurs in the presence of the agent. If UCP2-mediated cellular function occurs in the cells which lack a functional UCP2 gene in the presence of the agent, then the agent is a UCP2 mimic.
In another embodiment, the method of identifying an agent which mimics UCP2 activity comprises introducing the agent into a transgenic non-human mammal which lacks a functional UCP2 gene and determining whether a UCP2-mediated cellular function (one or more) occurs in the presence of the agent. If UCP2-mediated cellular function occurs in the transgenic non-human mammal which lacks a functional UCP2 gene in the presence of the agent, then the agent is a UCP2 mimic.
In the method of identifying a UCP2 mimic, examples of a (one or more) UCP2-mediated cellular function includes, for example, altered insulin/glucose homeostasis and responsiveness to glucose stimulation, altered mitochondrial activity, decreased responsiveness to glucose-stimulation, decreased level of insulin production, decreased ambient fed-state serum insulin levels combined with a decreased glucose level.
The present invention also relates to methods of treatment or prevention of conditions (e.g., hyperglycemia) or diseases (e.g., type 2 diabetes) associated with aberrant UCP2 function (e.g., negative regulation or uncoupling activity). In one embodiment the invention provides a method of increasing insulin production in an individual comprising administering to the individual an agent which inhibits UCP2 activity. In another embodiment, the invention provides a method of decreasing blood glucose in an individual comprising administering to the individual an agent which inhibits UCP2 activity. The invention further provides a method of treating diabetes (e.g., type 2 diabetes; non-insulin dependent diabetes mellitus) in an individual comprising administering to the individual an agent which inhibits UCP2 activity. An alternative embodiment of this aspect of the invention further provides a method of modulating mammalian mitochondrial activity (e.g., respiration rates and inner mitochondrial membrane potential). In one embodiment, the invention provides a method of coupling mitochondria (e.g., cellular respiration and ATP production) in an individual comprising administering to the individual an agent which inhibits UCP2 activity. Alternatively, the present invention relates to a method of enhancing the uncoupling of cellular respiration and ATP production in an individual comprising administering to the individual an agent which enhances (activates, induces) UCP2 activity.
Thus, the invention provides a source of cells and animals useful for elucidating the function of UCP2 in intact animals whose genomes comprise a wild-type UCP2 gene. In addition, Applicants have discovered that UCP2 negatively regulates insulin production in animals and uncouples mitochondrial respiration. Further aspects of the invention provide a method for the identification of agents (e.g., therapeutic agents) which inhibit UCP2 activity; and a method of treating diseases or conditions associated with UCP2 function (e.g., negative regulation or uncoupling activity).